The lack of effective glioblastoma treatments remains a significant health problem and highlights the need for novel and innovative approaches. Immunotherapy is an appealing strategy because of the potential ability for immune cells to traffic to and destroy infiltrating tumor cells. For the past decade, our group and others have been testing active vaccination strategies, such as dendritic cells pulsed with tumor lysates or synthetic peptides to induce antitumor immunity in glioblastoma patients. However, our data, and that of other immunotherapeutic strategies for patients with cancer, suggest that the vast majority of tumor-specific T cells induced by this personalized, patient-specific immunotherapy do NOT recognize well-characterized, known antigens. In order to design the most effective immunotherapeutic strategies for glioblastoma, we believe that it is critical to understand which antigens tumor-specific T cells recognize in this disease. Recent information suggests that patients mounting immune responses after immunotherapy can recognize novel neoantigens created by tumor-specific mutations. Our hypothesis is that glioblastoma patients treated with autologous tumor lysate-pulsed DC vaccination will mount anti-tumor immune responses against specific mutations in their individual tumor. Furthermore, we hypothesize that patients with extended survival will have mounted more diverse anti-tumor immune responses to such neoantigens. To test this, we propose to perform exome sequencing on patient tumor specimens to identify nonsynonymous mutations in glioblastoma patients treated with DC vaccination. We will then screen and identify candidate epitopes for glioma-specific T cell recognition, and finally evaluate which neoepitope- specific T cells are preferentially expanded following autologous tumor lysate-pulsed DC vaccination. We will also design a set of parallel pre-clinical studies in our orthotopic murine glioma model. We will characterize nonsynonymous mutations in two murine glioma cell lines, and subsequently identify dominant neoantigens recognized by murine glioma-specific T cells. To expand this, we will then vaccinate mice with identified glioma-specific neoantigens and evaluate which antigens confer effective anti-tumor immunity to mice bearing intracranial gliomas. This project could potentially be transformative, as a better understanding of the relevant neoantigens in malignant glioma could dramatically alter immunotherapy for this deadly disease. The studies proposed herein could have important implications for the development of personalized cancer vaccines in glioblastoma patients.